JOURNAL TRANSCRIPT

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Ph.D Thesis

Gene Polymorhisms of the Interleukin-10, Angiotensin-Converting Ensyme and Transforming Growth Factor-β 1 Genes in Granulomatous Disorders, Pulmonary Sarcoidosis and Wegener’s Granulomatosis By Gabriella Muraközy M.D. Semmelweis University School of Ph.D. Studies Clinical Medicine Respiratory Diseases Semmelweis University Faculty of Pulmonology Supervisor: Prof. Pál Magyar M.D., Ph.D, D.Sc. Opponents: László Kalabay M.D.,Ph.D Zoltán Balikó M.D., Ph.D Board of examiners: Prof. Endre Vastag M.D., Ph.D László Kalabay M.D., Ph.D Mária Szilassy M.D., Ph.D

Budapest 2005

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CONTENTS: CONTENTS.................................................................................................................2. ABBREVIATIONS...................................................................................................4. 1. 1.1.

INTRODUCTION.......................................................................................6. The cytokine network and cytokine gene polymorphisms in sarcoidosis......6.

1.1.1. The role of TGF-β1 in sarcoidosis........................................................................8. 1.1.2. The role of IL-10 in sarcoidosis..........................................................................9. 1.2.

Immunmodulatory cytokines in Wegener’s granulomatosis.........................9.

1.2.1. The role of IL-10 in Wegener’s granulomatosis.................................................9. 1.2.2. The role of TGF-β1 in Wegener’s granulomatosis.............................................10. 1.3.

Gene polymorphisms of the cytokine genes...................................................11.

1.3.1. IL-10 gene polymorphisms.................................................................................11. 1.3.2. TGF-β1 gene polymorphisms.............................................................................11. 1.3.3. ACE gene polymorphisms..................................................................................11.

2.

AIM OF THE STUDY...............................................................................12.

3.

MATERIALS AND METHODS............................................................13.

3.1.

Study population...............................................................................................13.

3.1.1. Sarcoidosis patients............................................................................................13. 3.1.2. Study population for Wegener’s granulomatosis...............................................15. 3.1.3. Control group......................................................................................................15. 3.2.

DNA extraction.................................................................................................15.

3.3.

Typing of the TGF-β1 codon 25.......................................................................16.

3.4.

Typing of the IL-10 gene promoter region.....................................................17.

3.5.

Typing of the ACE gene...................................................................................19.

3.6.

Statistical analysis.............................................................................................21.

4.

RESULTS.......................................................................................................22.

4.1.

The genotype and allele frequencies of the TGF-β1 and IL-10 PMs in sarcoidosis patients.......................................................................................22.

4.2.

The genotype and allele frequencies of the ACE PM in Wegener’s

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granulomatosis patients and controls.............................................................23. 4.3.

The genotype and allele frequencies of the TGF-β1 PM in Wegener’s granulomatosis patients and controls.............................................................23.

4.4.

The genotype and allele frequencies of the IL-10 PM in Wegener’s granulomatosis patients and controls.............................................................24.

5.

DISCUSSION...............................................................................................25.

5.1.

TGF-β 1 and IL-10 gene PMs in the sarcoidosis group..................................25.

5.2.

TGF-β 1, ACE and IL-10 gene PMs in the Wegener’s granulomatosis Group.................................................................................................................27.

6.

CONCLUDING REMARKS..................................................................33.

7.

ACKNOWLEDGMENTS.......................................................................34.

8.

REFERENCES............................................................................................35.

9.

ORIGINAL COMMUNICATIONS....................................................41.

9.1.

Original articles associated with the thesis....................................................41.

9.2.

Original articles not associated with the thesis.............................................41.

9.3.

Oral presentation associated with the thesis.................................................41.

9.4.

Oral presentation not associated with the thesis...........................................42.

9.5.

Abstracts...........................................................................................................45.

10.

SUMMARY...................................................................................................47.

11.

ÖSSZEFOGLALÁS...................................................................................48.

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ABBREVIATIONS: A

adenin

AAV

ANCA-associated vasculitis

ANCA

anti-neutrophil cytoplasmic autoantibodies

Arg

arginin

BAL

bronchoalveolar lavage fluid

Bp

base pair

C

cytosin

ConA

concanavalin A

D

deletion

DLco

diffusing capacity

DNA

deoxyribonucleoside triphosphates

dNTPs

deoxyribonucleic acid

FEV1

forced exspiratory volume in 1 second

G

guanin

HLA

human leukocyte antigen

IL-10

interleukin-10

IL-2

interleukin-2

IL-6

interleukin-6

IFNγ

interferon gamma

I

insertion

MIP2

migration inhibitor protein 2

MPA

microscopic polyangiitis

MPO

myeloperoxidase

PBMC

peripheral blood mononuclear cells

PCR

polymerase chain reaction

PM

polymorphism

Pro

prolin

PR3

proteinase 3

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Raw

airway resistance

SACE

Serum angiotensin-converting ensyme

sIL-2R

soluble interleukin-2 receptor

SNPs

single nucleotid polymorphisms

SSOP

sequens specific oligonucleotid primer

TGF-β1

transforming growth factor-β1

Th1

T helper 1 cell

Th2

T helper 2 cell

TLC

total lung capacity

TNFα

tumor necrosis factor alfa

VC

vital capacity

WG

Wegener’s granulomatosis

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INTRODUCTION 1.1. The cytokine network and cytokine gene polymorphisms in sarcoidosis Sarcoidosis is a chronic systemic granulomatous disease of unknown origin in

which environmental exposures are belived to interact with genetic factors in determining the pattern of sarcoidosis presentation, progression and prognosis. The consensus is that the disease results from an exagregated cellular immune respons ( acquired, inherited or both ), disease activity is accompanied by mononuclear cell infiltrates and granuloma formation. The most common organs involved being the lung and lymph nodes, although almost any organ can be affected, including the eyes, heart, skin and central nervous system. The course and progression of the disease are correlated with the mode of onset, where an acute onset accompanied by fever, arthralgia and erythema nodosum ( named Löfgren syndrome ) usually heralds a self limited course with spontaneous resolution. An insidious onset may be followed by chronic disease with relentless progressive fibrosis. For an individual patient, however, only time and close observation will provide a specific answer regarding the disease course, making an appropiate treatment at the time of disease diagnosis difficult. Although many attempts to find prognostic markers for a better assessment of sarcoidosis and to ameliorate treatment of patients have been made, a reliable marker is still laeking. The granuloma formation in sarcoidisis is characterised by an accumulation of CD4+ T lymphocytes and mononuclear phagocytes [1] . Sarcoidosis is associated with an increase in the number of alveolar T-cells and a shift to an increase in CD4+ cells within these cells can be seen. The interaction of the activated cells require mechanisms of signal transduction, the cytokine network of which has been analysed in detail as the required methods have been available since the early 1980s. In this complet network, the effect of an individual cytokines varies with the grade of activation of the target cell, the presence of other cytokines in the local microenvironment and the ability of the target cells to produce arachidonic acid metabolites. In the course of sarcoid alveolitis an abundance of cytokines, soluble cytokine receptors and soluble adhesion molecules capable of attracting and activating immune cells and of inducing and maintaining

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granuloma are released by alveolar macrophages, T-cells and epithelial cells. Previous studies analysing lung inflammatory cells recovered by bronchoalveolar lavage (BAL) revealed, that T cells exhibit a T helper type 1 (Th1) cytokine pattern [2] . A great number of cytokines is produced by alveolar cells during inflammatory reactions of sarcoidosis. Alveolar macrophages release IL-1 and TNF-α, alveolar T-cells activated by alveolar macrophage-derived IL-12 release IL-2, IL-16, INF-γ and other cytokines which attract mononuclear cells to the alveoli, activate these cells and induce their proliferation and differentiation. Alveolar T-cells and macrophages activate each other and there is evidence that the cytokine pattern of the alveolitis determines the course of the disease. In this complex network the balance between pro- and anti-inflammatory cytokines might be of major importance. Elevated levels of IL-2, IL-8 and TNF-α and MIPa were found in patient with progressive disease, which demonstrate that these immunopathological processes determine the clinical course of sarcoidosis. Moreover, by measuring alveolar macrophage TNF-α release it may be possible to identify subgroups of sarcoidosis patients who will suffer from progression in the near future. At present the most extensively investigated deactivating cytokines are IL-10 and TGFβ1. IL-10 inhibits cytokine production as well as proliferation of human monocytes and T-cells. TGF-β1 is an immunomodulator, exhibiting pro- and anti-inflammatory activities and inhibiting the development of Th1 cells. Evidence for genetic predisposition for sarcoidosis has long been assumed due to the prevalence of the disease of the different ethnic groups and due to familiar clustering. It is belived that a supposed genetic susceptibility is most probably conferred by more than one immune regulatory gene, which act synergistically on disease risk. Numerous studies have been performed to clarify this aspect and especially, the highly polymorphic HLA locus has been investigated intensivly. Apart from contributing to susceptibility to sarcoidosis, it is also probable, that genetic factors may be important in defining the pattern of disease presentation and progression as well as its overall prognosis. For example the association of the B8-DR3 genotype or TNFA2 gene has been associated with good prognosis in a number of studies.

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For other diseases like multiple sclerosis modifying roles of cytokine gene PM have been discovered for different alleles of the interleukin-4 gene, although its detailed mechanism remains elusive [3] .

1.1.1. The role of TGF-β1 in sarcoidosis TGF-β belongs to a family of ubiquitous regulatory proteins that are crucial for cell growth, cell differentiation and regulation of extracellular matrix production. TGFβ is produced by several cell types present in the lung, for example alveolar macrophages, T-cells, bronchial epithelial cells and type II alveolar epithelial cells. There is growing evidence confirming the role of TGF-β as an immunmodulator, inhibiting the differentation of Th1 cells and exhibiting both pro- and anti-inflammatory activities [4]. There is strong evidence that TGF-β induces the production of extracellulare matrix and that high TGF-β production is associated with several fibrotic disease. On the other hand, there are hints of immunosuppressive capabilities of TGF-β inhibiting T-cell activation and downregulating activated peritoneal macrophages. In a previous study TGF-β could be detected in BAL supernatants of sarcoidosis patients with active disease and spontaneous remission within 6 months after the investigation whereas TGF-β levels of patients with indication for therapy or suffering from chronic disease did not differ from controls. Furthermore a strong and significant negative correlation was found between IL-2 and TGF-β1 production by BAL cells. This suggest an inhibitory role of TGF on the IL-2 production of T-cells. In sarcoidosis, the release of TGF-β by alveolar macrophages (AM) seems to be indicative for an ongoing immune response that results in cessation of the inflammatory processes and subsequently in spontaneous resolution of the disease [5] . Whether TGF-β1 is the key cytokine in downregulating the alveolitis or whether it acts together with other, still unknown mediators require further investigation.

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1.1.2. The role of IL-10 in sarcoidosis IL-10 is a Th2 type cytokine, that exerts an anti-inflammatory activity by inhibiting Th1 type responses and monocyte function. Although several investigators made efforts to detect this molecule, its presence in the BAL cells supernatant of sarcoidosis could not be established [5] , but indirect results regarding its gene transcription by BAL cells have been obtained [6] and elevated serum levels have been recorded in sarcoidosis patients [7]. IL-10 and TGF-β1 are cytokines with strong immunoregulatory capabilities, gene polymorphisms (PMs) in the promoter regions of these cytokines are associated with altered transcription rates, leading to genetically determinated high or low producers [8, 9]. The consequence could be imbalances in the cytokine network and subsequent immunopathological changes could promote either spontaneous resolution or chronic progression of sarcoidosis. Therefore, these genes were interesting candidates of disease modifying genes, defined as inherited genetic variations that lead to a quantitative difference in the development of the disease phenotypes.

1.2. Immunmodulatory cytokines in Wegener’s granulomatosis 1.2.1. The role of IL-10 in Wegener’s granulomatosis Despite the unknown origin of the disease the analysis of the infiltrating inflammatory cells and their products are of major importance in understanding the pathogenesis of WG. As previously shown the granuloma formation in WG is characterized by CD4+ T cells which exhibit increased secretion of interferon-γ (IFNγ) but not of IL-4 or IL-10 leading to a Th1 response [10]. Moreover it was demonstrated for peripheral blood mononuclear cells (PBMC) isolated from patients with WG that IL10 can prevent the development of a Th1 response, in a dose-dependent blocking effect on IFNγ production [11].

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1.2.2. The role of TGF-β 1 in Wegener’s granulomatosis Due to the known strong association between antineutrophil cytoplasmic antibody (ANCA) and WG, proteinase-3 (PR3) a lysosomal protein, which is the main target autoantigen of ANCA, is of great interest [12]. PR3 is normally located in the azurophil granules of human neutrophils. TGF-β1 and some other proinflammatory cytokines such as IL-6 and tumor necrosis factor-α seem to be capable of translocating PR3 to the cell surface making the autoantigen (PR3) accessible to the corresponding autoantibody (ANCA) [13]. Therefore, TGF-β1 should be included into the hypothetical model of the development of WG. According to this model, the interaction between PR3 and ANCA results in degranulation of polymorphonuclear cells, release of lysosomal protease, generation of oxygen radicals leading to endothelial cell injury [14]. ( See in Figure 1. ) Figure 1. The role of TGF-β1 in the pathogenesis of Wegener’s granulomatosis PMN

ANCA TGF-β1

PR3

IL-6

PR3

TNF-α ICAM-1

LFA-1

Endothelial cell (EC)

activated

EC-PMN adhesion

PR3-ANCA-IC

O2ANCA activated PMN cells

PMN and EC lysis

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1.3. Polymorphisms of the cytokine genes 1.3.1. IL-10 gene polymorphism The IL-10 gene is located on chromosome 1q31 [15]. Screening the promoter region for point mutation has revealed a G/A exchange at position -1082 from the transcription start site, which was found to be associated with different levels of IL-10 production by ConA stimulated peripheral blood mononuclear cells in healthy individuals [9]. Individuals, negativ for IL-10 allele A at position -1082 show significantly higher IL-10 production after ConA stimulation compared to IL-10 A allele positive individuals, which means, that allele G is associated with a significantly higher IL-10 production. 1.3.2. TGF-β 1 gene polymorphism The TGF-β1 gene is located on chromosome 19q13, and a biallelic G/C PM in codon 25 is significantly associated with different levels of TGF-β1 production. Healthy individuals with genotype GG produce higher levels of TGF-β1 [16] than those with CG. Due to a low frequency of allele C individuals, homozygous for CC constitute less than 1 % of the population and could not be tested until now [8]. 1.3.3.

ACE gene polymorphism

Serum ACE levels are known to be elevated in sarcoidosis, a granulomatous disorder of unknown origin as well as in chronic beryllium disease and some other autoimmune disorders [17]. In sarcoidosis it has been shown that serum ACE activity reflects the granuloma burden [18], and a deletion/insertion PM in intron 16 of the ACE gene, was shown to be responsible for a great percent of the variability in serum ACE activity [19]. This PM is inherited in a mendelian manner yielding to 3 genotypes. A Finnish study observed an association between DD genotype and phenotype of chronic course in sarcoidosis [20]. In parallel, another group showed, that

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in chronic beryllium disease the DD genotype associated with an earlier onset of the disease and a lower beryllium lymphocyte proliferation response compared to non-DD genotypes [21]. Thus, ACE PM seems to modify the phenotype of various diseases suggesting a general involvement in the inflammatory response of granulomatous disorders. 2.

AIM OF THE STUDY

1. The aim of this study was to evaluate the biallelic G/C polymorphism in codon 25 of the TGF-β1 gene in sarcoidosis, Wegener’s granulomatosis and healthy persons to get information whether the genotype frequencies of the named PMs differ in sarcoidosis or in WG in comparison to healthy persons. 2. Furthermore to evaluate the biallelic A/G PM at position -1082 (relative to the first translational site) of the IL-10 gene in sarcoidosis, Wegener’s granulomatosis and healthy persons to get information whether the genotype frequencies of the named PMs differ in sarcoidosis or in WG in comparison to healthy persons. 3. To determinate the deletion/insertion PM in intron 16 of the ACE gene in Wegener’s granulomatosis and healthy persons to get information whether the genotype frequencies of the named PMs differ in WG in comparison to healthy persons. 4. Furthermore I investigated the genotype frequencies of the named TGF-β1 and IL-10 PMs in distinct phenotypes of sarcoidosis to get information about the influence of the PMs to the course of the sarcoidosis.

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3. MATERIALS AND METHODS 3.1. Study populations 3.1.1. Sarcoidosis patients: A total of 51 unrelated patients (21 female, 30 male, mean age: 41.8 ± 14.1 years) suffering from sarcoidosis were included in the study. The diagnosis of sarcoidosis was established on previously defined criteria, including transbronchial lung biopsy [22]. The patients were retrospectivly grouped according to clinical criteria and the course of the disease. Group I (n=21) was defined as patients with no indication for therapy at the time of diagnosis and after 6 month a reevaluation of the disease showed spontaneous remission. Group II (n=11) was defined as patients with indication for therapy at the time of diagnosis. The patients received corticosteroids for 6 to 12 months and after 1 year of follow-up the status of the disease was reevaluated and judged as either stable or regressive without any sign of relapse. Group III (n=19) was defined as recalcitrant disease exhibiting signs of inflammation, progressive symptoms, reduction in pulmonary function or progressive chest X-ray findings, not allowing to taper off corticosteroids for two years. To evaluate the disease activity and indication for therapy chest X-ray, lung function test, messaurement of serum parameters such as angiotensin-converting enzyme (sACE), neopterin, soluble IL-2 receptor (sIL-2R) levels, and analysis of BAL fluid cells were performed as peviously described [23-25]. The gender and age distribution between our sarcoidosis groups was similar, however the mean age of patients in group III was slightly higher, although the difference was not significant. At the time of diagnosis the given serological parameters showed elevated levels as a sign of active inflammation in all three groups. The lung function parameters of group I were within the normal range, whereas group II and to a greater extend group III presented with significantly decreased lung function parameters, especially the diffusion capacity of the lung (Dlco). These damages are in line with the greater damage of the lung tissue in these patients. The BAL fluid cytology in all groups reflected a typically T4-helper lymphocyte alveolitis concordant with the diagnosis of sarcoidosis, however in the chronic-progressive group the neutrophil percentage was significantly elevated. The characteristics of the sarcoid groups are shown in table 1.

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Table 1. Characteristic of the sarcoidosis patients (mean values ± SEM) *p